One of the purposes of the proposed investigation is to continue our study into the ways in which lanthanide ions can be used to probe protein structure. The varied spectral and magnetic properties of these ions can be used to determine binding sites and distances between inhibitors and the metal ions. They can also be used to detect conformational changes in the protein due to a variety of perturbants introduced into solution via the alterations in the measured distances. A determination of the stereochemistry of the substrate or inhibitor bound to the active site will also be attempted by measurement of distances from different parts of the inhibitors chosen to one or more metal ions. Thermolysin, a calcium containing thermostable protein will be the principal subject of these investigations. The similarity in size and chemical behavior of the calcium and lanthanide ions should allow the results obtained in the protein-lanthanide system to be extended to protein-calcium systems which play an active role in the chemistry of the body, e.g., blood clotting, muscle contraction, etc. A direct investigation of these systems using the calcium ion is severely hampered by the absence of any readily measurable spectral properties of the calcium ion. Two additional protein systems will be studied. Ribonuclease A and B will be a trial system to investigate the possibility of inserting a metal ion binding site via an EDTA-boronate derivatives into a carbohydrate containing system. This will enable us to extend the fluorescence and nmr techniques utilizing lanthanide ions, as well as other metal ions, to proteins which do not normally bind metal ion tightly. We are also going to investigate the effect of a variety of molecules and ions that bind to human serum albumin, causing a large conformational change. Several different metal ions, including lanthanide ions, in addition to barbital and other drugs bind to human serum albumin and may promote this conformational change.